PROTECTION FROM THE ACUTE CISPLATININDUCED NEPHROTOXICITY BY Eruca Sativa
SEED OIL IN RATS
Authors: Al-Thamir, S. N. K.a, Al-Shalah, H. H.b and Bairam, A. F. H.c
a Department of Pharmacology- College of Pharmacy/ University of Babylon
b Department of Clinical Biochemistry- College of Medicine/ University of Babylon
c Department of Pharmacology and Toxicology- College of Pharmacy/ University of
Kufa
Abstract
Introduction: The usefulness of cisplatin, a potent anti-tumor, is limited by
its ability to induce nephrotoxicity. The cellular changes leading to this
toxicity are suggested to be mediated by increased free radicals generation
and lipid peroxidation.
Aim of the study: To investigate the protective properties of eruca sativa
seed oil on cisplatin-induced nephrotoxicity in rats using biochemical and
histopa- thological approaches.
Material and methods: 35 healthy male Swiss albino rats were used. They
were divided into 5 groups (7 animals in each group and all animals supplied
with standard food during the experiment): control group (1 ml/kg of
normal saline i.p. once daily for 14 days), cisplatin treated group (7.5 mg/kg,
i.p. single dose), eruca sativa seed oil treated group (1 ml/kg, orally, once
1
daily for 14 days), eruca sativa seed oil (1 ml/kg) plus cisplatin treated group
(orally, once daily for 7 days before and after cisplatin injection), and eruca
sativa seed oil (1 ml/kg) plus cisplatin treated group (orally, once daily for
14 days before and after cisplatin injection). Blood samples were collected
and used to determine the serum urea, creatinine and total antioxidant status
(TAS) levels. kidneys were removed and prepared for histopathological
examinations.
Results: In eruca sativa seed oil plus cisplatin treated groups serum urea and
creatinine were significantly lower than those of cisplatin-treated group,
While serum TAS was increase. These changes occurred in a dosedependant manner (eruca sativa seed oil). Histopathological examinations
showed a massive damage in the proximal tubules in cisplatin-treated group.
No damage was observed in eruca sativa seed oil treated groups.
Conclusion: These data show that eruca sativa seed oil can provide a
protective effect against Acute cisplatin-induced nephrotoxicity. This
protective effect of eruca sativa seed oil may be related to the antioxidant
status on the kidney.
Key wards: cisplatin, eruca sativa seed oil, acute nephrotoxicity, total
antioxidant status (TAS).
‫ألخالصة‬
‫ فائدة السزبالتين كعقار مضاد للسرطان محدودة بسبب تأثيره السمي للما اليليمال الرايمرا‬:‫المقدمة‬
‫الخلويا اليؤديا إلا هذه السييا سببها زيادة ف تيوين الجذور الحرة وأكسدة الدهون‬
‫ تقييم الرأثير الوقائ لزيت بذور الجرجير للا الرمأثير السمي للسمزبالتين للما‬: ‫الهدف من الدراسة‬
‫اليليا ف الجرذان باسرخدام اخربارا كيييائيا حياتيا وإجراء الفحص النسيج ل‬
2
‫الحيوانات‪ ,‬المواد وطرق العمل‪ :‬خيسا وثالثين ذكرا بالاا من الجرذان البيض السويسريا اسمرخدمت‬
‫ف هذه الدراسمال همذه الحيواتما قسميت بعميا لعموائ إلما خيسما مجمامي (سمبعا جمرذان فم كما‬
‫مجيولا وجيي الحيواتا ألطيت غمذاء طبيعم قياسم طموات الرجربماح‪ :‬الحيواتما فم اليجيولما‬
‫أألولا ألطيت ماء ملح طبيع ‪1‬ما‪/‬كام لن طريق البيريرون مرة واحدة يوميما ليمدة أربعما لعمر‬
‫يوم واُلربر مجيولما ألسميطرة أألوليمال الحيواتما فم اليجيولما الحاتيما ُحقنمت بيمادة السمزبالتين‬
‫جرلا واحدة ‪7‬ل‪ 5‬ملام‪/‬كام ف الرجويف ألبطن (البيريرونحل ف حمين إن الحيواتما فم اليجيولما‬
‫الحالحا ألطيت زيت بذور الجرجير ‪1‬ما‪/‬كام لن طريق الفم مرة واحدة يوميا ليدة أربعا لعمر يمومل‬
‫الحيواتا ف اليجيولا الرابعا ألطيت زيت بمذور الجرجيمر ‪1‬مما‪/‬كام لمن طريمق الفمم ممرة يوميما‬
‫لن طريق الفمح ليدة أربعا لعر يوم سبعا أيام قبا وسبعا أيام بعد جرلا السزبالتينل الحيواتا ف‬
‫اليجيولا الخامسا ألطيت زيت بذور الجرجير ‪1‬ما‪/‬كام لن طريق الفم مرة يوميا لن طريق الفمح‬
‫ليدة ثيان ولعرون يوم‪ ,‬أربعا لعر يوم قبا وأربعا لعر يوم بعمد جرلما السمزبالتينل لينما المدم‬
‫جيعممت واسممرخدمت لقيمماش اليؤلممرا الراليمما‪ :‬مؤلممرا وهيفمما اليلمما وهم يوريمما الممدم وكريمماتينين‬
‫اليصا وفحص مؤلر األكسدة وهو ( ت أي أش ح ف اليصال كيا تم ليا الفحص النسيج لليليال‬
‫النتائج‪ :‬ف اليجامي اليعاملا بالسزبالتين باإلضافا إلا زيت بذور الجرجير كمان مسمروا اليوريما و‬
‫اليرياتنين ف اليصا بصورة واضحا اقا من مسروياتها ف اليجيولا اليعاملا بالسمزبالتين لوحمده‪,‬‬
‫بينيا ان مسروا (أي ت أشح ف اليصا ازدادلهذه الرايرا حدثت بطريقا تعريد للما جرلما زيمت‬
‫بذور الجرجيرحل الفحص النسيج بين وجود ضرر كبير ف النبيبا اليلويا اليلرويما فم اليجيولما‬
‫اليعاملمما بالسممزبالتين لوحممدهلكيا اتمما لممير هنالممي ضممرر يالحممم ف م اليجممامي اليعاملمما بالسممزبالتين‬
‫باإلضافا إلا زيت بذور الجرجيرل‬
‫االستنتاج‪ :‬هذه البياتا‬
‫تبين إن زيت بذور الجرجير ييرلي تأثير وقائ‬
‫ضد الرأثير السي‬
‫للسزبالتين للا اليليا وهذا الرأثير الوقائ يعزا إلا الفعاليا اليضادة لألكسدة للا اليليال‬
‫‪Introduction‬‬
‫‪Cisplatin is an antineoplastic drug, administered to cancer patients as a‬‬
‫‪sterile solution. It is widely used for the treatment of several human‬‬
‫‪malignancies (as standard component of treatment regimens) including head‬‬
‫‪3‬‬
and neck cancers [1], testicular cancer [2], small-cell [3] and non-small cell
lung cancer [4], ovarian cancer [5], cervical cancer [6] and bladder cancer
[7]. The main dose-limiting side effect of cisplatin is nephrotoxicity [8]
Cisplatin nephrotoxicity can present in a number of ways. However, the
most serious and one of the more common presentations is the acute renal
failure which occurs in 25–35% of patients [9]. Several investigators have
hypothesized the oxidative stress mechanism of cisplatin induced
nephrotoxicity that is related to depletion of the antioxidant defense system
[10] and to inhibitory effect of cisplatin on antioxidant enzymes activities
[11]. On the other hand, eruca sativa seed (ESS) oil contains a range of
health-promoting phytochemicals including carotenoids, vitamin C, fibers,
flavonoids, and glucosinolates (GLs) and their breakbown products, e.g.
isothiocyanates (as sulforaphane) [12]. Sulforaphane has several biological
activities
including
anticarcinogenic,
antifungal,
antibacterial
and
antioxidant effects [20].
Animals, materials and methods
Animals: A total of 35 adult male Albino Swiss rats aged 16 - 24 weeks
with weight of (170 – 255g), were obtained from Animal Resource Center,
the Institute of Embryo Research and Treatment of Infertility, Al-Nahrain
University. The animals were apparently healthy and they were housed in
individual cages, at temperature controlled environment (25±5 ◦C) with
ambient humidity. Lights were maintained on a 12h light/dark cycle. The
rats received standard chow diet with water (ad libitum). Rats in the study
were maintained in accordance with the Guide for the Care and Use of
Laboratory Animals USA 1996 [13].
4
Study design:
After 4 weeks acclimatization period, the animals were randomly separated
into 5 groups (7 rats in each group):
i. Normal saline (N.S) treated group: all rats of this group received normal
saline (1 ml/kg, i.p., once daily) for 14 days.
ii. Cisplatin treated group: all rats of this group received cisplatin (7.5
mg/kg i.p.) single dose.
iii. ESS oil treated group: all rats of this group were given ESS oil (1 ml/kg,
orally, once daily) for 14 days.
iv. ESS oil treated group (1 ml/kg) plus cisplatin: all rats of this group were
given ESS oil (orally) once daily for 7 days before and 7 days after
cisplatin injection.
v. ESS oil treated group (1 ml/kg) plus cisplatin: all rats of this group were
given ESS oil (orally) once daily for 14 days before and 14 days after
cisplatin injection.
Materials and methods: Cisplatin 50mg/dl vial (Ebewe pharma, Austria).
ESS oil was freshly obtained from dr. Maher Salman college of agriculture
AL-Kufa university. The oil extracted from the seeds according to Stahl,
(1969) method by using Soxhlet extractor apparatus and petroleum spirit as a
solvent [21]. ESS oil was given by oral gavage.
All other chemicals were of the highest available analytical grade. After
24hr from the last injection of all treatments, the rats were anesthetized with
phenobarbital (50 mg/kg s.c.). Blood samples (3mls) were obtained from
each rat by cardiac puncture using disposable syringe. Sera were obtained
via centrifugation at 3000 rpm for 10 minutes and preserved at -20 °C until
5
determination of parameters of serum urea, creatinine and TAS by
commercially available colorimetric kits. Rats abdomen was opened through
a midline incision and the kidneys were quickly removed and fixed in 10%
formalin for histopathological examination.
Statistical analysis of data: Statistical analyses were performed using SPSS
version 18 (GrraphPad, ISI software, Philadelphia, PA, USA, 1993)
computer program. Data are expressed as means ± standard deviation
(M±SD). Multiple comparisons were done using one way ANOVA
Bonferroni test. The (p<0.05) level of probability was chosen as a criterion
for the lowest level of significance.
Results
Effects of cisplatin and Eruca Sativa seed oil treatment on serum urea
and creatinine concentrations :
According to Figures 1 and 2, serum urea and creatinine levels were
significantly (p<0.01) higher in cisplatin-treated group than the controls.
ESS oil produced a significant (p<0.01) and dose dependent reduction in the
urea and creatinine concentrations when compared with the cisplatin treated
group.
6
Figure 1. Concentration of serum urea in different treated groups. Data was shown as
mean ± S.D. ** = p<0.01 as compared with cisplatin-treated group (n = 7). Oil 1wp = oil
1 week (before and after cisplatin injection) protection group, oil 2 wp = oil 2 weeks
(before and after cisplatin injection) protection group.
Figure 2. Concentration of serum creatinine in different treated groups. Data was shown
as mean ± S.D. ** = p<0.01 as compared with cisplatin- treated group (n = 7).
7
Effects of cisplatin and Eruca Sativa seed oil treatment on serum TAS :
According to Figures 3, serum TAS were significantly (p<0.01) lower in
cisplatin-treated group than the controls. ESS oil produced a significant
(p<0.01) and dose dependent elevation in the serum TAS when compared
with the cisplatin treated group.
Figure 3. Concentration of serum total antioxidant status in different treated groups. Data
was shown as mean ± S.D. ** = p<0.01 as compared with cisplatin- treated group (n = 7).
Kidney histology:
All parts of kidney showed normal appearance in the control group (Figure
4 A). On the other hand, rats treated with cisplatin showed tubular
degeneration which varied according to kidney zone. Coagulative necrosis
was observed in the tubular lining epithelium at the cortex and
corticomedullary junction, the protein casts are seen in the lumen of
proximal tubules and loss of the epithelial brush-border (Figure 4 B1 and
8
B2). Coagulative necrosis is an irreversible grade of necrotic cell death and
tubular cells could not regenerate after removal of toxic insult. Treatment
with ESS oil alone didn't show any significant histopathological changes
compared to the control group (Figure 4 C). The treatment with ESS oil 1
ml/kg for one week and two weeks before and after cisplatin injection
showed nephroprotective and antinecrotic effect of the histopathological
changes induced by cisplatin in a dose dependant manner (Figures 4 D and
E) respectively.
A
B1
B2
C
9
D
E
Figure (4) Light microscopy of renal tissue stained with hematoxylin and
eosin stain in different treated groups (high magnification power, × 400);
(A) Control group, renal tubules show normal appearance. (B1) Cisplatin
treated group, Proximal tubules show coagulative necrosis in the corticomedulary junction (arrow). (B2) Cisplatin treated group, the tubular
epithelial cell casts are in the lumen of the proximal tubules (arrow). (C)
ESS oil treated group (oil alone), show no histopathological changes
compared with the control group. (D) ESS oil plus cisplatin treated group
(14 days protection), almost all renal proximal tubules are intact. (E) ESS oil
plus cisplatin treated group (28 days protection), all of the renal proximal
tubules in the cortex are intact and show no histopathological changes
compared with the control group.
Discussion
Cisplatin is one of the most effective anticancer agents. It is used to treat
solid tumors such as testicular, bladder, ovarian, breast and lung cancers
[14]. The present study indicated that ESS oil dramatically protected the
cisplatin-induced in vivo nephrotoxicity in rat.
10
In the present study, a single dose of cisplatin (7.5 mg/kg, i.p.) induced
nephrotoxicity as revealed from the increase in serum urea, creatinine levels
and decrease serum TAS as well as by histopathological findings. These
changes occurred acutely (24 hours) after cisplatin injection into rats. These
results are consistent with those experimentally reported by [15,16].
The main mechanism of cisplatin induced nephrotoxicity is oxidative stress
through lipid peroxidation and free radical generation in the tubular cells
causing tubular cell necrosis as reported by [17]. The proposed mechanisms
by which cisplatin causes oxidative stress are:
a. Depletion of the antioxidant defense system [10].
b. Inhibitory effect of cisplatin on antioxidant enzymes activities as catalase
(CAT), glutathione peroxidase (GSH-Px) and super oxide dismutase (SOD),
[11].
The increase in free radical generation and the decrease in antioxidant
defense system results in an increase in serum and renal lipid peroxidation
that lead to decrease the level of TAS production in serum and renal tissue.
The results of this study confirm the nephro-protective activity of ESS oil,
When the rats received ESS oil (1ml/kg for 14 days for a group and 28 days
for the other group) the serum urea and creatinine concentrations are
significantly lower than that in cisplatin administered group, while the serum
TAS is more than that in cisplatin administered group. These results are in
agreement with [18, 19]. The possible factors behind the protective effects of
ESS oil is related to its contents of bioactive substances. Glucosinolates
were found to have several biological activities including anticarcinogenic,
antifungal, antibacterial
plus
their antioxidant
action. The major
glucosinolate in ESS is Erucin, which is potentially capable of protecting
cells against oxidative stress via three mechanisms: (i) induction of phase II
11
enzymes, (ii) scavenging hydrogen peroxide and alkyl hydroperoxides
accumulated in cells and peripheral blood and (iii) acting as a precursor of
sulforaphene, a potent inducer detoxifying electrophiles and increase cellular
antioxidant defenses [22].
The mechanism of protective activity of sulforaphene is the induction of
phase II detoxifying enzymes [e.g., glutathione transferases (GSTs, EC
2.5.1.18), NAD(P)H: quinone reductase (QR, EC 1.6.99.2), epoxide
hydrolase, and heme oxygenase], which are important in the detoxification
of electrophiles [23].
Conclusions: ESS oil is able to protect the kidneys against cisplatininduced acute renal failure. But, before a conclusive statement on potential
usefulness of ESS oil as adjunct to the cisplatin therapy, there is a need for
further studies including human trials.
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